Unraveling the complexity of anti-doping analysis: reassessing meldonium detection and doping verdicts in a case study.

BACKGROUND: The precision and accuracy of mass spectrometry (MS) made it a fundamental tool in anti-doping analysis. High-resolution (HR) mass spectrometers significantly improved compound identification. This study systematically analyzes data from an athlete (Subject 1) who tested positive for meldonium and compares it with data from a healthy volunteer (Subject 2) to examine the correctness of the doping verdict. CASE PRESENTATION: The documentation related to Subject 1 was thoroughly processed and analyzed. A study involving a volunteer (Subject 2) replicated Subject 1 regimen and urine sample collection for data alignment with anti-doping results, with Subject 2 reporting not using meldonium. The anti-doping agency's analysis of Subject 1 showed the presence of meldonium at a concentration close to the established cut-off level. However, a closer examination revealed that one specific ion, crucial for meldonium identification, was absent from the mass spectra. Analyzing Subject 2 data, using the same methodology, the absence of the specific ion was confirmed, even though the volunteer did not consume meldonium. The European directive and the method that was validated and cited by the anti-doping agency identified meldonium on at least four specific ions, whereas the anti-doping analysis used only three ions. This discrepancy compromises the specificity of meldonium identification. CONCLUSIONS: To enhance the analytical methodology, two strategic interventions are suggested: adjusting the meldonium cut-off value and expanding the analysis to include meldonium metabolites. By addressing these avenues, the precision of meldonium detection and doping verdicts can be improved. In conclusion, this study challenges the anti-doping agency's verdict and prompts a reevaluation of meldonium detection methodologies in anti-doping measures.

Meldonium residues in milk: A possible scenario for inadvertent doping in sports?

Lately, the veterinary drug Emidonol® has been discussed as a possible scenario for inadvertent doping in sports. Emidonol® is approved for use in livestock breeding, exhibiting antihypoxic and weak sedative effects. The veterinary drug rapidly dissociates into meldonium, a substance prohibited in sports, and is excreted largely in its unchanged form into urine. To investigate if residues of meldonium in edible produce may result in adverse analytical findings in sports drug testing, a pilot study was conducted with three volunteers consuming a single dose of 100 ml meldonium-spiked milk at a concentration of 500 ng/ml (Study 1), and multiple doses of 100 ml of meldonium-spiked milk (500 ng/ml) on five consecutive days (Study 2). In the single dose study, urinary meldonium concentrations peaked between 2 and 6 h post-administration with maximum values of 7.5 ng/ml, whereas maximum meldonium concentrations of 18.6 ng/ml were determined after multiple doses 4 h post-administration. All samples were analyzed using an established and validated protocol based on HILIC-HRMS/MS.

Fluorescence chemosensing of meldonium using a cross-reactive sensor array.

In this paper, we report a fluorescent sensor array approach for the urinary detection of a prohibited substance in sports, meldonium. Four chemosensors with ethidium bromide scaffolds were employed in this method. The interaction between meldonium and chemosensors was investigated by different techniques, such as ultraviolet-visible absorption and fluorescence spectroscopy, nuclear magnetic resonance, and mass spectrometry. Molecular dynamics simulation was also used to elucidate and support the interaction mechanisms between meldonium and the chemosensors. Differential responses obtained from the sensor array enabled the qualitative and quantitative analyses of meldonium with low error values. This method was able to detect and quantify meldonium at the nM level, fulfilling the requirements of minimum performance defined by the World Anti-Doping Agency.

Meldonium long-term excretion period and pharmacokinetics in blood and urine of healthy athlete volunteers.

Meldonium is a metabolic drug whose inclusion in the 2016 List of Prohibited Substances and Methods followed the analysis of data collected under the 2015 World Anti-Doping Agency Monitoring Program. In the early months of 2016, anti-doping laboratories reported an unusually high number of cases in which urine samples contained high concentrations of meldonium. Consequently, the meldonium excretion period in healthy athletes and the substance's long-term urine and blood (plasma) pharmacokinetics became central questions for the anti-doping community to address, to ensure appropriate assessment of the scientific and medical situation, and also fair treatment of athletes from a result management and legal standpoint. At the present time, data on meldonium pharmacokinetics is limited to a few studies, with no known data available on long-term excretion of high oral doses. The primary objective of this open-label study was to determine long-term urine and plasma pharmacokinetic parameters of meldonium in healthy volunteers. Study design included single and repeated functional load testing and assessment of L-carnitine administration on meldonium excretion and pharmacokinetics. Thirty-two volunteers were equally divided into two groups receiving either 1.0 g or 2.0 g of oral meldonium daily for 3 weeks. The study found meldonium takes several days to attain a steady state in blood and displays an elimination period over several months after cessation of treatment. Moreover, findings demonstrate that the daily dose, periodicity and duration of treatment with meldonium are the most important factors to consider in calculating the substance's elimination and complete body clearance.

Urinary excretion studies of meldonium after multidose parenteral application.

Meldonium is a drug exhibiting cardioprotective and anti-ischemic effects. Due to its potential performance-enhancing benefit in sports, meldonium was added to the World Anti-Doping Agency list of prohibited substances in 2016. Since then, a high number of adverse analytical findings reported on meldonium has questioned meldonium`s detection time in urine. Hence, the objective of the current study was to characterize the pharmacokinetic urinary excretion pattern of meldonium when administered as multiple intravenous injections. Three injections of 250 mg meldonium were given over a time period of five days to six healthy volunteers and urine samples were collected for eight months after the last injection of the drug. For the quantification of meldonium in urine, a liquid chromatography-tandem mass spectrometry method was fully validated according to the World Anti-Doping Agency guidelines in terms of specificity, matrix interferences, intra- and inter-day precision, accuracy, carry-over, robustness, linearity, limit of detection, and limit of quantification. The assay was successfully applied to the pharmacokinetic study. A three-compartment model was found to best describe the pharmacokinetics of meldonium with average alpha, beta, and gamma half-lives of 1.4 h, 9.4 h, and 655 h, respectively. The detection time in urine varied between 94 and 162 days.

The atypical excretion profile of meldonium: Comparison of urinary detection windows after single- and multiple-dose application in healthy volunteers.

Following a one-year monitoring program providing unequivocal analytical evidence for a high prevalence in international elite sports, meldonium has been included in the World Anti-Doping Agency's (WADA) list of prohibited substances that came into effect on 1 January 2016. Despite of the polar and hydrophilic nature of the molecule, an unusual long detection window was observed in pilot elimination studies. Consequently, in the present study, urinary excretion profiles after single-dose (5 volunteers, 1×500mg) and multiple-dose oral application (5 volunteers; 2×500mg/day for 6days) were determined in order to facilitate the result management concerning meldonium findings in doping controls. Particularly the option to differentiate between recent use and tapering concentrations was studied. Urinary meldonium concentrations were determined using an analytical approach based on hydrophilic interaction liquid chromatography and high resolution tandem mass spectrometry. The study corroborates the hypothesis of a non-linear, dose-depended and biphasic excretion profile after oral application of meldonium and demonstrates that urinary detection windows are of considerable extent with up to 65 and 117days (concentrations>LOQ of 10ng/mL) following single- and multiple-dose applications, respectively.

Story behind meldonium-from pharmacology to performance enhancement: a narrative review.

Recent reports from the World Anti-Doping Agency (WADA) indicate an alarming prevalence in the use of meldonium among elite athletes. Therefore, in January 2016, meldonium was added to WADA's prohibited list after being monitored since 2015. Meldonium has been shown to have beneficial effects in cardiovascular, neurological and metabolic diseases due to its anti-ischaemic and cardioprotective properties, which are ascribed mainly to its inhibition of ß-oxidation and its activation of glycolysis. Despite its widespread use, there are only a few clinical studies or clinical trials available. Meldonium is registered in most Baltic countries and is easily accessible through the internet with no serious adverse effects reported by the manufacturer so far. Among athletes, meldonium is used with the purpose of increasing recovery rate or exercise performance. The benefit of taking meldonium in view of performance enhancement in athletes is quite speculative and is discussed without sound scientific evidence. This narrative review provides a detailed overview of the drug meldonium, focusing on the main topics pharmacology and biochemical actions, clinical applications, pharmacokinetics, methods of detection and potential for performance enhancement in athletes.

Rapid determination of meldonium in urine samples by capillary electrophoresis with capacitively coupled contactless conductivity detection.

Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) was employed for fast determination of meldonium (MEL) in urine samples. Background electrolyte consisting of 2M acetic acid (pH 2.3) was used for separation of MEL from cationic compounds present in urine samples and the overall analysis time was less than 4min per sample. Direct injection of urine samples was possible after 1:9 dilution with deionized water. This simple sample pretreatment was sufficient to eliminate possible matrix effects on CE performance and allowed for precise and sensitive determination of free MEL in urine. Excellent linearity (r2≥0.9998) was obtained for two concentration ranges, 0.02-4µgmL-1 and 2-200µgmL-1, by simply changing injection time from 10 to 2s without the need for additional dilution of urine samples. Limit of detection was 0.015µgmL-1 and average recoveries from urine samples spiked at 0.02-123.5µgmL-1 MEL ranged from 97.6-99.9%. Repeatability of migration times and peak areas was better than 0.35% and 4.2% for intraday and 0.95% and 4.7% for interday measurements, respectively. The above reported data proved good applicability of the CE-C4D method to determination of various MEL concentrations in urine samples and good long-term performance of the analytical system. The method might be particularly useful in analyses of large batches of samples for initial testing of MEL-positive vs. MEL-negative urine samples.

Meldonium use by athletes at the Baku 2015 European Games.

BACKGROUND: The aim of this report was to estimate the prevalence of meldonium use in athletes competing in the Baku 2015 European Games to contribute to the surveillance of substances on the 2015 World Anti-Doping Agency (WADA) Monitoring Program. Meldonium is reported to be used by athletes to potentially enhance personal performance and shorten the recovery period after physical activity. METHODS: Three sources of data were reviewed to determine the prevalence of meldonium use during the Games including: (1) athlete self-reported declarations of drug and supplement use; (2) declarations from National Olympic Committee medical teams of the list of medicines that they imported into Azerbaijan as part of their stock of drugs for administration; (3) results from the antidoping laboratories reporting the detection of meldonium. RESULTS: Meldonium was declared as imported into Azerbaijan by 2 of 50 National Olympic Committee medical teams at the Games, but athletes from 6 countries declared the use of meldonium. Only 23 of the 662 (3.5%) athletes tested from 8 to 28 June 2015 declared the personal use of meldonium, which included 13 competition winners. However, 66 of the total 762 (8.7%) athlete urine samples analysed during the Games and during precompetition tested positive for meldonium. Meldonium use was detected in athletes competing in 15 of the 21 sports during the Games. CONCLUSIONS: This study highlights the widespread and inappropriate use and prescribing of this prescription drug in a generally healthy athlete population. Subsequent to these findings, WADA has included meldonium as a prohibited substance on the 2016 List of Prohibited Substances.

Mildronate (Meldonium) in professional sports - monitoring doping control urine samples using hydrophilic interaction liquid chromatography - high resolution/high accuracy mass spectrometry.

To date, substances such as Mildronate (Meldonium) are not on the radar of anti-doping laboratories as the compound is not explicitly classified as prohibited. However, the anti-ischemic drug Mildronate demonstrates an increase in endurance performance of athletes, improved rehabilitation after exercise, protection against stress, and enhanced activations of central nervous system (CNS) functions. In the present study, the existing evidence of Mildronate's usage in sport, which is arguably not (exclusively) based on medicinal reasons, is corroborated by unequivocal analytical data allowing the estimation of the prevalence and extent of misuse in professional sports. Such data are vital to support decision-making processes, particularly regarding the ban on drugs in sport. Due to the growing body of evidence (black market products and athlete statements) concerning its misuse in sport, adequate test methods for the reliable identification of Mildronate are required, especially since the substance has been added to the 2015 World Anti-Doping Agency (WADA) monitoring program. In the present study, two approaches were established using an in-house synthesized labelled internal standard (Mildronate-D3 ). One aimed at the implementation of the analyte into routine doping control screening methods to enable its monitoring at the lowest possible additional workload for the laboratory, and another that is appropriate for the peculiar specifics of the analyte, allowing the unequivocal confirmation of findings using hydrophilic interaction liquid chromatography-high resolution/high accuracy mass spectrometry (HILIC-HRMS). Here, according to applicable regulations in sports drug testing, a full qualitative validation was conducted. The assay demonstrated good specificity, robustness (rRT=0.3%), precision (intra-day: 7.0-8.4%; inter-day: 9.9-12.9%), excellent linearity (R>0.99) and an adequate lower limit of detection (<10 ng/mL).

Determination of mildronate in human plasma and urine by liquid chromatography-tandem mass spectrometry.

A sensitive and selective analytical method based on liquid chromatography-triple-quadrupole mass spectrometer has been developed to determine mildronate in human plasma and urine. The aim of this work was to find a valid method to study the pharmacokinetic profiles of mildronate in humans. Mildronate is a heart protection medicine, a carnitine's structural analogue, so levocarnitine was used as an internal standard for quantification. Under the electrospray ionization source positive ion mode, calibration curves with good linearities (r=0.9998 for plasma sample and r=0.9999 for urine sample) were obtained in the range of 1.0-20,000 ng ml(-1) for mildronate. The detection limit was 1 ng ml(-1). Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of mildronate in humans, and to the best of our knowledge, this is the first report on LC-MS-MS analysis of mildronate in plasma and urine.

Analysis of carnitine and acylcarnitines in urine by capillary electrophoresis.

A capillary electrophoresis method is described for the simultaneous analysis of carnitine and short-chain acylcarnitines in aqueous standard solutions and urine samples. Samples were worked up using silica gel extraction and derivatization with 4'-bromophenacyl trifluoromethanesulfonate. Separation was performed in less than 8 min using a binary buffer system containing phosphate/phosphoric acid and sodium dodecyl sulfate. 3-(2,2,2-Trimethylhydrazinium)propionate (mildronate) was used as an internal standard. The method was developed with aqueous standard solutions and then applied successfully to spiked and unspiked human urine samples. The limit of detection for both carnitine and acetylcamitine is 3 microM.